Background: RG7787 is a next generation recombinant immunotoxin (RIT) developed by the Pastan Lab (NCI/ LMB) in collaboration with Roche. This RIT binds to the cancer antigen mesothelin (MSLN) that is expressed on at least 70% of pancreatic adenocarcinomas and delivers a potent bacterial toxin to the cell cytosol. The toxin, a molecularly engineered variant of Pseudomonas exotoxin A, kills cells by irreversibly modifying a critical enzyme in the protein synthesis pathway resulting in a halt in the cell's ability to produce new proteins. This insult triggers apoptosis in many cell types. AIM 1. Determine whether the combination of RG7787 and NAB-paclitaxel improves anti-tumor efficacy in pre-clinical models. The Pastan lab had previously demonstrated that combination of RG7787 with the chemotherapy drug paclitaxel resulted in synergistic improvement of anti-tumor efficacy in a mouse model of pancreatic cancer (Hollevoet et al, Mol Canc Ther, 2014). We would like to take advantage of this favorable interaction when giving RITs to patients, but paclitaxel is an ineffective drug for treating pancreatic cancer and would not be a good choice to use in clinical trials. NAB-paclitaxel is a closely related drug of the taxane family that does have efficacy in pancreatic cancer and is approved for use in this disease. We tested the combination of NAB-paclitaxel and RG7787 in vivo in a mouse model of pancreatic cancer. While each drug halted tumor growth individually, when given together they produced durable complete responses in most of the mice treated. In order to better understand how the combination works, we developed an in vitro model that mimicked the in vivo results. Using this model, we discovered that this favorable interaction occurred in additional tumor cell lines and the schedule of the two drugs necessary to produce improved killing. We found that the toxin portion of RG7787 was required for the boosted effect and that addition of the taxane did not change how well a given dose of RG7787 could inhibit proteins synthesis. Conclusions: RG7787 and NAB-paclitaxel do interact favorably in pre-clinical models and the combination warrants testing in the clinic. Further study will be required in order to understand how the two drugs interact to produce this effect. AIM 2. Determine whether delivery of RG7787 to tumors can be improved by co-administration of an anti-fibrotic drug. Pancreatic adenocarcinoma is a paucicellular tumor that contains a large volume of dense surrounding stroma that inhibits delivery of anti-cancer therapeutics, provides cytokines that help the tumor cells survive under stress, and help to suppress anti-tumor immune activation. Pancreatic cancers co-opt pro-fibrotic signaling cascades that are physiologically part of the normal wound healing process in order to produce this desmoplasia. Studies from the Pastan lab have indicated that delivery of RG7787 to pancreatic tumors growing in mice is heterogeneous. While RG7787 levels are high enough in some cells to kill them, levels in many other cells are sufficient only to cause a temporary growth arrest (E. Mason-Osann et al, Sci Reports, 2015). We examined whether co-administration of RG7787 with an FDA approved anti-fibrotic drug could improve anti-tumor efficacy in athymic nude mice bearing xenografted pancreatic tumors. We found that although the anti-fibrotic could alter signaling to the stromal cells, it did not improve anti-tumor activity over treatment with RG7787 alone. We have initiated a collaboration with Perwez Hussian (NCI/LHC) to see if the anti-fibrotic may have effect in a model system that more closely resembles human pancreatic tumors. Conclusions: Therapy with this anti-fibrotic agent does not increase RG7787 efficacy in an immunosuppressed subcutaneous xenograft model of pancreatic cancer. AIM 3. Determine whether RG7787 or other mesothelin-targeted RITs can boost the effect of immune activating drugs like checkpoint inhibitors. Pancreatic adenocarcinoma produces an immunosuppressive microenvironment. Killing tumor cells with oncolytic viruses or administering anti-tumor vaccines can cause immune activation. Combining these treatments with immune checkpoint inhibition has been demonstrated to produce anti-tumor immune responses in pre-clinical models of pancreatic cancer. We hypothesize that since RG7787 uses a bacterial toxin to kill tumor cells, it may also induce immune activation within the pancreatic cancer microenvironment that could be leveraged to induce an anti-tumor immune response in combination with immune checkpoint inhibition. We have developed and characterized a new syngeneic mouse model that would allow us to test the activity of mesothelin-targeted RITs in combination with checkpoint inhibitors. We are also collaborating with the CAPR group at Frederick (Terry VanDyke & Serguei Kozlov) to develop additional models that would be suitable for helping us to answer this important question. AIM 4. Evaluate efficacy of mesothelin-targeted RITs in pancreatic cancer patients. At least 70% of pancreatic adenocarcinomas make the surface antigen mesothelin, making these tumors good targets for mesothelin-targeted therapies. Raffit Hassan (NCI/ TGIB) has demonstrated that the mesothelin-targeted RIT SS1P can produce durable major responses in heavily pre-treated patients with malignant mesothelioma (Hassan et al, Sci Trans Med, 2013), but this combination has not been tested in pancreatic cancer. As stated above, RG7787 has promising pre-clinical activity in mesothelioma and pancreatic cancer. In collaboration with Dr. Hassan, his clinical protocol testing the SS1P/ pentostatin + cyclophosphamide regimen has been amended to include pancreatic cancer patients. This amendment was approved within the last month, allowing us to enroll pancreatic cancer patients on the trial. A Phase I study of RG7787 (renamed Ro6927005) was begun by Roche (12/2015) as a multi-center international trial that included NCI as the only U.S. site (NCI PI, Hassan; Lead AI, Alewine). We treated one patient at NCI on this study.